ABSTRACT
Salmonella spp. are estimated to cause 1.2 million cases of human foodborne illness each year in the United States, and pigs can often be asymptomatically colonized with Salmonella spp. (>50% of farms). Recent reports state that 18.3% of Salmonella enterica serovar Typhimurium isolates are resistant to ≥3 antimicrobial classes, and multidrug-resistant (MDR) strains are associated with an increased hospitalization rate and other complications. Chlortetracycline is commonly used in swine production to prevent/treat various diseases; therefore, chlortetracycline treatment of pigs unknowingly colonized with MDR Salmonella may have collateral effects on Salmonella spp. (and other gut bacteria). In this study, we determined the effect of in-feed chlortetracycline (400 g/ton) on shedding and colonization of pigs challenged with the MDR S. Typhimurium strain DT104 (n = 11/group). We also assessed the impact on the fecal microbiota over the 12-day experimental period and on the ileum, cecum, and tonsil microbiota at 7 days postinoculation (dpi). In MDR S. Typhimurium-inoculated pigs, chlortetracycline administration significantly increased fecal shedding at 2 dpi (+1.4 log10 CFU/g; P < 0.001) and enhanced tonsil colonization (+3.1 log10 CFU/g; P < 0.001). There were few major alterations detected in the gut or tonsillar microbiota of pigs treated with MDR S. Typhimurium and/or chlortetracycline. The tonsillar transcriptome was largely unaffected despite increased colonization by MDR S. Typhimurium following inoculation of the chlortetracycline-treated pigs. These results highlight the idea that chlortetracycline administration can enhance shedding and colonization of MDR S. Typhimurium in pigs, which could increase the risk of environmental dissemination of MDR Salmonella strains.
IMPORTANCE Salmonella spp. are an important cause of foodborne illness in North America, and pork products are associated with sporadic cases and outbreaks of human salmonellosis. Isolates of Salmonella may be resistant to multiple antibiotics, and infections with multidrug-resistant (MDR) Salmonella spp. are more difficult to treat, leading to increased hospitalization rates. Swine operations commonly use antimicrobials, such as chlortetracycline, to prevent/treat infections, which may have collateral effects on pig microbial populations. Recently, we demonstrated that chlortetracycline induces the expression of genes associated with pathogenesis and invasion in MDR Salmonella enterica serovar Typhimurium in vitro. In our current study, we show increased tonsillar colonization and fecal shedding of the MDR S. Typhimurium strain DT104 from pigs administered chlortetracycline. Therefore, pigs unknowingly colonized with multidrug-resistant Salmonella spp. and receiving chlortetracycline for an unrelated infection may be at a greater risk for disseminating MDR Salmonella spp. to other pigs and to humans through environmental or pork product contamination.
INTRODUCTION
Nontyphoidal Salmonella spp. are among the most common causes of human foodborne illness in North America and the leading cause of foodborne illness-related hospitalization and death (1, 2). Salmonellosis can occur following the consumption of contaminated pork, and swine can be an important reservoir of Salmonella spp., with an isolation rate varying between 50 and 100% of farms testing positive for this foodborne pathogen (3–7). In the latest report by the National Antimicrobial Resistance Monitoring System (NARMS), 18.3% of human Salmonella enterica serovar Typhimurium isolates were classified as multidrug resistant (MDR) due to resistance to at least three classes of antimicrobials (8). In humans, multidrug-resistant nontyphoidal Salmonella infections are associated with higher rates of hospitalization and bacteremia than are antimicrobial-susceptible strains (9).
Chlortetracycline has been used in agriculture for nearly 70 years and is administered to pigs to prevent or control disease, including in the treatment of bacterial enteritis and respiratory infections, at a therapeutic dosage of 400 g/ton (363 mg/kg) of feed for up to 14 days (10, 11). Chlortetracycline has low bioavailability (6%) in pigs when given orally (12) and is excreted via fecal and renal routes (13). Thus, the gut microbiome is exposed to relatively high concentrations of chlortetracycline following in-feed administration. In one of the earliest studies of chlortetracycline use (100 g/ton) on MDR S. Typhimurium shedding in swine, Williams and others (14) recorded an increase in the concentration of Salmonella spp. in fecal samples and a longer duration of fecal shedding in treated pigs. Likewise, Delsol et al. (13) noted an increase in Salmonella fecal shedding for up to 7 days postinoculation (dpi) in pigs that were inoculated with S. Typhimurium DT104 and given 15 mg chlortetracycline/kg of feed. Chlortetracycline administered at 120 μg/ml in drinking water has also been demonstrated to increase Salmonella concentrations in the ceca of chickens challenged with MDR S. Typhimurium compared to those inoculated with a sensitive S. Typhimurium strain (15). Therefore, exposure to chlortetracycline clearly confers a competitive advantage to MDR Salmonella strains in the gastrointestinal tract of animals, at least temporarily. However, these studies did not look at changes in tissue colonization or the gut microbiota within a week of MDR Salmonella inoculation, nor did they use the 400-g/ton therapeutic regimen of chlortetracycline as commonly used in the United States.
Previously, we demonstrated that chlortetracycline induces the expression of virulence genes and their subsequent phenotypes in MDR Salmonella strains in vitro (16). Genes involved in pathogenesis and attachment that are not typically expressed in vitro were markedly upregulated after chlortetracycline exposure, with cellular invasion being enhanced in some strains. Therefore, in the present study, we investigated the in vivo effect of chlortetracycline on Salmonella colonization, fecal shedding, the host tonsil transcriptome, and alterations to the microbiota in pigs inoculated with the MDR S. Typhimurium DT104 strain. With simultaneous chlortetracycline administration and MDR Salmonella inoculation, we hypothesized that chlortetracycline-induced changes in the gastrointestinal microbiota would result in higher levels of pathogen colonization and fecal shedding.
RESULTS
Changes in white blood cell populations in response to Salmonella inoculation.The concentrations of band neutrophils, lymphocytes, monocytes, and neutrophils, as well as white blood cell counts, were significantly increased at one or more sampling times in the Salmonella-inoculated pigs (nonmedicated feed [i.e., without chlortetracycline {CTC}] and MDR Salmonella inoculation [−CTC/+SAL] and medicated feed and MDR Salmonella inoculation [+CTC/+SAL] groups) compared with pigs in the two noninoculated groups (nonmedicated feed and no MDR Salmonella inoculation [−CTC/−SAL] and medicated feed and no MDR Salmonella inoculation [+CTC/−SAL]), indicating an immunological response to the Salmonella inoculation (see Fig. S1 in the supplemental material). The addition of chlortetracycline to the diet did not affect the white blood cell populations in the Salmonella-inoculated pigs.
Pig health and fecal parameters.The growth rates of the four treatment groups did not differ significantly from −5 to 0, 0 to 7, or −5 to 7 dpi (Table S1). Pigs grew faster from −5 to 0 dpi than from 0 to 7 dpi, which might be attributed to more frequent handling and sampling during the 0 to 7 dpi period that may have negatively affected appetite. No consistent changes were observed in fecal moisture content in relation to chlortetracycline supplementation or SB 403 inoculation (Fig. S2A). The pigs in the +CTC/+SAL group had the highest rectal temperature at 1, 2, and 4 dpi, although it was only significantly elevated at 2 dpi compared to that in the +CTC/−SAL pigs (Fig. S2B). Similarly, pigs in the −CTC/+SAL group that were inoculated with strain SB 403 had a significantly increased rectal temperature at 2 dpi compared to the –CTC/−SAL pigs that were mock inoculated. Consistent with the transient increase in the swine rectal temperature, the serum gamma interferon (IFN-γ) levels were significantly increased (P < 0.05) at 2 dpi compared to those at 0 dpi for both the +CTC/+SAL (334.2 and 13.51 pg/ml, respectively) and −CTC/+SAL (356.7 and 17.77 pg/ml, respectively) groups. A statistical difference was not observed for serum IFN-γ levels comparing 0 or 2 dpi between the +CTC/+SAL and −CTC/+SAL groups.
Salmonella fecal shedding and tissue colonization.All pigs were Salmonella negative prior to 0 dpi, and all noninoculated pigs remained fecal negative for Salmonella spp. from 0 to 7 dpi. The concentration of Salmonella spp. in the feces was relatively stable from 1 to 7 dpi in the −CTC/+SAL pigs and did not differ by sampling time (Fig. 1A). However, the pigs in the +CTC/+SAL cohort shed significantly higher levels of Salmonella spp. at 2 dpi than did the −CTC/+SAL pigs (+1.4 log10 CFU/g; P < 0.001). Samples from the ileocecal lymph nodes, distal ileum, cecal contents, and cecal mucosa at 7 dpi all had similar concentrations of Salmonella spp. and were not significantly different between the −CTC/+SAL and +CTC/+SAL groups (Fig. 1B; P > 0.05). Notably, the tonsil tissue of +CTC/+SAL pigs had significantly higher concentrations (3.1 log10 CFU/g) of Salmonella spp. than −CTC/+SAL pigs (P < 0.001) at 7 dpi. In addition, a significant difference (P = 0.035) in colonization of the tonsils was observed with 5 of the 11 pigs in the −CTC/+SAL group being culture negative for Salmonella, whereas all tonsil samples from the +CTC/+SAL pigs were Salmonella positive.
Multidrug-resistant Salmonella Typhimurium fecal shedding (A) and tissue colonization (B) by treatment group. Tissue samples were taken from pigs euthanized at 7 days postinoculation. Error bars indicate the standard error of the mean. *** indicates significantly different means (P < 0.001). –CTC, received nonmedicated feed; +CTC, received in-feed chlortetracycline (CTC) at 400 g/ton of feed; −SAL, unchallenged pigs; +SAL, challenged with Salmonella Typhimurium SB 403. ICLN, ileocecal lymph node.
Pig gut and tonsillar microbiota.The fecal microbiota of all treatment groups changed very little throughout the experiment, even after chlortetracycline administration and SB 403 inoculation (Fig. 2). From 1 to 7 dpi, the largest factor affecting the fecal microbiota structure was the individual pig itself (R2 = 0.40; P < 0.001), followed by sampling time (R2 = 0.06; P < 0.001), chlortetracycline administration (R2 = 0.03; P < 0.001), and SB 403 inoculation (R2 = 0.02; P < 0.001). Overall, the treatment groups were most dissimilar from each other at 1 dpi (Fig. S3). MDR S. Typhimurium inoculation had no major biological effect on the tonsillar, ileal mucosal, cecal mucosal, or cecal content microbiota (Fig. 3; R2 < 0.05). Similarly, in-feed chlortetracycline had only minor effects on the microbiota (R2 < 0.08). Thus, it appears that the treatment groups shared many of the same taxa in similar abundances throughout the experimental period. The fact that pigs grouped most strongly by individual animal also shows the lack of treatment effect on the fecal microbiota.
Principal-coordinate analysis (PCoA) plot of Bray-Curtis dissimilarities of the fecal microbiota by treatment group at 0, 1, 2, 3, and 7 days postinoculation (dpi). −CTC, received nonmedicated feed; +CTC, received in-feed chlortetracycline at 400 g/ton of feed; −SAL, unchallenged pigs; +SAL, challenged with Salmonella Typhimurium SB 403. dpi, days postinoculation. The percentages of variation explained by the principal coordinates are indicated on the axes.
Pairwise Bray-Curtis dissimilarities between treatment groups for each tissue. Error bars represent ± standard error of the mean, and different lowercase letters within each tissue indicate significantly different means (P < 0.05). Higher values indicate groups with microbiota that are more dissimilar. The box in the box plots indicates the interquartile range (IQR) (middle 50% of the data), the middle line represents the median value, and the whiskers represents 1.5 times the IQR. Colored dots indicate outliers.
Although variability within each treatment group by sampling day was observed, Salmonella inoculation and chlortetracycline supplementation together tended to reduce the richness (number of operational taxonomic units [OTUs]) of the fecal microbiota, as −CTC/−SAL pigs had significantly more OTUs than those in the +CTC/+SAL group at 4 and 7 dpi (P < 0.05; Table S2). At 4 dpi, the −CTC/−SAL pigs also had significantly higher Shannon diversity values than did +CTC/+SAL pigs (P < 0.05). Compared to their litters, the fecal microbiota of the sows was more rich and diverse than that of any of the pig groups at all sampling times. Among the samples taken during necropsy at 7 dpi, −CTC/−SAL pigs had significantly more OTUs in their cecal and ileal mucosal microbiota than did the other three treatment groups (P < 0.05; Table 1). As expected, the microbiota of the cecal contents and mucosa, as well as the feces, had greater diversity and OTU richness than did the ileal mucosal and tonsillar samples.
Number of OTUs and Shannon diversity index values for the tonsils, ileal mucosal, cecal contents, and cecal mucosal microbiotaa
Based on the significant difference observed in Salmonella tonsil colonization at 7 dpi and fecal shedding at 2 dpi between the −CTC/+SAL and +CTC/+SAL groups, we identified differentially abundant OTUs between these two groups (Table S3). Five OTUs were differentially abundant in the fecal microbiota of the −CTC/+SAL and +CTC/+SAL pigs at 2 dpi, including three OTUs that were classified as Lactobacillus or Succinivibrio spp. and were depleted in the +CTC/+SAL pigs. However, all of these differentially abundant OTUs were less than 1.0% in relative abundance and highly variable. In the tonsillar microbiota, only a single OTU was differentially abundant between the –CTC/+SAL and +CTC/+SAL groups, an OTU classified as Alloprevotella that was below 1% relative abundance in the –CTC/+SAL group and not detected in the +CTC/+SAL pigs.
The cecal contents, cecal mucosa, and fecal microbiota shared many of the same abundant genera, including Alloprevotella, Lactobacillus, Megasphaera, Phascolarctobacterium, Prevotella, Streptococcus, and Subdoligranulum (Fig. S4 to S6). The ileal mucosa and the tonsils both had a unique microbiota in comparison with those found in the lower gastrointestinal tract (Fig. S7 and S8). The ileal mucosal microbiota was largely dominated by members of Lactobacillus and Streptococcus and the tonsillar microbiota by Actinobacillus, Bacteroides, and Veillonella. Notably, Campylobacter was among the 10 most abundant genera in both the ileal mucosa and the tonsils. Salmonella was only detected by 16S rRNA gene sequencing in the tonsils of the pigs inoculated with SB 403 (9/22) and not in any of the other sample types (data not shown).
Transcriptomic changes in the tonsils.The effect of chlortetracycline and SB 403 inoculation on host transcription in the tonsils was determined, since a large and significant difference in Salmonella colonization between the −CTC/+SAL and +CTC/+SAL pigs was observed in this tissue. Only one gene, ALDH3A1, which codes for an aldehyde dehydrogenase, was found to be differentially expressed between these two groups at 7 dpi (Table S4; false-discovery rate [FDR] < 0.10). Compared to the −CTC/−SAL group, no differentially expressed genes in either the −CTC/+SAL or +CTC/+SAL group were detected. Additionally, samples did not cluster by treatment group, and there was considerable interindividual variability in the transcriptome (Fig. 4). Most of the genes that were highly expressed in the tonsils were housekeeping genes, and in total, the expression of 13,175 genes among all samples from all treatment groups was detected (Table S5).
Principal-component analysis (PCA) of the regularized log2-transformed gene expression counts for each tonsillar RNA sample in each treatment group at 7 days postinoculation. The percentages of variation explained by the principal components are indicated on the axes.
DISCUSSION
Recently, we reported that in vitro exposure of MDR S. Typhimurium strains to chlortetracycline induces the upregulation of genes associated with attachment, invasion, and pathogenesis, including genes not otherwise expressed in culture (16). Resistance to chlortetracycline in SB 403 is mediated through tet(G), an efflux protein-encoding gene (17) located on Salmonella genomic island-1 (SGI-1), along with the resistance genes aadA2, blaPSE, and floR (18, 19). Consequently, these genes are also being selected for in this strain during chlortetracycline exposure. In the current study, we determined the effect of concurrent chlortetracycline administration on MDR S. Typhimurium shedding and colonization in postweaned pigs. We observed a transient increase in Salmonella fecal shedding at 2 dpi and enhanced colonization of the tonsils at 7 dpi in pigs administered in-feed chlortetracycline at 400 g/ton. These findings are in agreement with those from other challenge studies that have described the tonsils as an important site for Salmonella colonization and persistence (20, 21). For swine naturally exposed to Salmonella spp. in the production environment, the quantity of Salmonella spp. shed in the feces is varied, and active shedding of Salmonella spp. by pigs is intermittent (7). However, periods of stress (feed withdrawal, transportation, etc.) can result in pathogen recrudescence with increased Salmonella shedding, including pigs where pathogen shedding was previously undetectable (22, 23).
Of six swine tissues, Salmonella spp. were reported to be most frequently isolated from the tonsils in finishing pigs at slaughter (24). However, Salmonella recovery from the tonsils at slaughter is potentially a combination of preexisting Salmonella colonization and tonsil contamination due to fecal exposure from actively shedding pigs during recent transport and lairage. Although the isolation of Salmonella spp. from tonsils following slaughter is a potential harbinger for the presence of the pathogen in other tissues, the role of contaminated tonsil tissue in adulteration of pork products is poorly understood. Wood et al. (21) analyzed 35 tissue types for up to 28 weeks following Salmonella inoculation and demonstrated that tonsils (93%) were most frequently colonized with the foodborne pathogen. Patterson et al. (23) suggested that the tonsil is a likely reservoir for Salmonella spp. during stress-induced recrudescence. In pigs administered chlortetracycline, we observed both a rise in the number of pigs with Salmonella-positive tonsils and an ∼3-log10 increase in tonsillar colonization by SB 403. Although fecal shedding of MDR Salmonella strains in our study was similar at 7 dpi in the presence or absence of chlortetracycline administration, greater colonization of the tonsils in pigs administered chlortetracycline may extend the duration of active pathogen shedding or enhance stress-induced Salmonella recrudescence.
The Salmonella Typhimurium DT104 530 strain (SB 403) was selected as the challenge strain in this study because it responded to chlortetracycline in vitro with elevated virulence gene expression (16). However, fecal shedding of SB 403 in the −CTC/+SAL pigs was lower than that in other challenge studies using S. Typhimurium strains that are not MDR (25–27), as were Salmonella SB 403 concentrations in the ileocecal lymph node (ICLN), cecum, and ileal mucosa (28, 29). The average rectal temperature for the SB 403-inoculated pigs also remained below 40°C (Fig. S2B), suggesting only a mild physiological response to this isolate. However, IFN-γ levels and white blood cell populations were elevated in the SB 403-inoculated pigs during one or more sampling times, indicating an immunological response to this strain. Isolates of DT104, including the 530 strain used in this study (18, 30), can have a lower rate of cellular invasion in vitro than that of sensitive isolates (31), which may explain the reduced clinical presentation and colonization.
The majority of the abundant genera in the cecal contents, cecal mucosa, and fecal microbiota were previously described as prevalent in the swine lower gastrointestinal tract (32). These genera included Alloprevotella, Blautia, Lactobacillus, Megasphaera, Phascolarctobacterium, and Streptococcus. One notable exception was Treponema, which had an overall relative abundance of only 0.29% among all pigs. This genus is typically one of the most abundant within the swine fecal microbiota (32), and this was reflected in its presence in the sows (13.4%). Although this finding suggests poor transfer of Treponema spp. from the sow to the piglet gut during the nursing phase, it should be noted that Treponema was among the abundant genera in the tonsil microbiota of several pigs (Fig. S8). Like most mammals, piglets acquire their initial microbiota through contact with their mother, coprophagy, and their environment (33). Consequently, differences in the fecal microbiota of the sows and their piglets could be related to age, diet, or the sanitary conditions under which the piglets were raised. Nevertheless, the growth rate was typical of pigs in this age range (34, 35) and, therefore, the gut microbiota differences observed between the piglets and their sows appeared to have no consequence on growth.
Although chlortetracycline had a larger effect on the swine microbiota than did SB 403 inoculation, the overall impact was still relatively low. This was an unexpected finding given the therapeutic dosage of chlortetracycline that was administered during the experiment. However, chlortetracycline is the oldest antimicrobial that continues to be used in food-producing animals, and it is possible that the most abundant bacteria of the swine gut microbiome are no longer sensitive to chlortetracycline. Indeed, the cultivable bacterial community from the swine gut can have a high rate of resistance (60% to 95%) to tetracycline/chlortetracycline even in the absence of chlortetracycline exposure (34, 36, 37). In further support of this, only in the ileal mucosal samples was the richness (number of OTUs) significantly higher in the control −CTC/−SAL pigs than in the +CTC/−SAL pigs.
At 7 dpi, the tonsillar transcriptome of all pigs was analyzed using high-throughput sequencing. Although >13,000 porcine genes were expressed among all of the tonsillar samples, only one gene was differentially expressed between the –CTC/+SAL and +CTC/+SAL groups despite the large difference in Salmonella colonization at 7 dpi. Moreover, no differentially expressed genes were observed between the −CTC/−SAL and +CTC/+SAL pigs. Only between +CTC/−SAL and the other groups of pigs was more than one differentially expressed gene identified. A targeted gene expression study by Volf et al. (38) using the tonsils of pigs challenged with Salmonella enterica serovar Enteritidis (5 dpi) detected changes in the expression of only 4 of 24 cytokine signaling genes, all of which were downregulated in the challenged pigs. Uthe et al. (39) showed that the expression of immune-related genes in the mesenteric lymph nodes of Salmonella Typhimurium-challenged pigs occurred within the first couple of days after challenge and waned by 7 dpi. It is therefore possible that differential gene expression between groups occurred prior to our transcriptional analysis at 7 dpi. Alternatively, the lack of a strong host response to Salmonella colonization of the tonsils may contribute to this tissue’s association with pathogen persistence in swine.
In summary, our assessment of the swine gut microbiota in response to MDR S. Typhimurium SB 403 challenge and chlortetracycline administration revealed that the pig gut microbiota remained relatively stable throughout the 12-day experiment, with only minor changes attributable to MDR S. Typhimurium and chlortetracycline. However, pigs inoculated with MDR S. Typhimurium SB 403 and given in-feed chlortetracycline had significantly higher levels of fecal shedding at 2 dpi and tonsil colonization at 7 dpi. These results may have implications for food safety and public health, as pigs treated with chlortetracycline and unknowingly colonized with MDR Salmonella could shed higher concentrations in their feces or harbor the foodborne pathogen longer in the tonsils, leading to an increased risk of environmental and pork product contamination.
MATERIALS AND METHODS
Bacterial strains.S. Typhimurium phage type DT104 strain 530 is resistant to at least five different antibiotic classes, and the MIC for chlortetracycline was 128 μg/ml (16, 18). This strain carries the tet(G) gene, which is encoded on Salmonella genomic island-1 (SGI-1) (30). A nalidixic acid-resistant derivative of S. Typhimurium strain 530 was constructed by transfer of a point mutation in the gyrA gene (C248T mutation resulting in Ser83Phe) of S. Typhimurium χ4232 into strain 530 by generalized transduction using P22 (40). Following selection on LB medium containing nalidixic acid, a single bacterial colony was isolated and stocked as BBS 1231. To minimize potential negative consequences on pathogenicity due to laboratory passage and genetic manipulation of BBS 1231, a pig was inoculated with BBS 1231, and a nalidixic acid-resistant S. Typhimurium isolate was recovered from the ileocecal lymph nodes (ICLNs) on xylose-lysine-tergitol 4 (XLT4) medium following necropsy and designated strain SB 403 (our unpublished data). The antimicrobial-resistant phenotype of SB 403 was confirmed to be consistent with that of BBS 1231 (resistant to ampicillin, tetracycline, streptomycin, chloramphenicol, and nalidixic acid). The virulent ICLN-isolated S. Typhimurium strain SB 403 was used for the swine experiment described herein.
Animals and experimental design.A total of 44 crossbred piglets from 5 sows that tested fecal negative for Salmonella spp. three times were included in the study at the National Animal Disease Center (NADC), Ames, IA, USA. Piglets were weaned at 21 days of age. At 6 weeks of age, pigs were blocked by sex, litter, and weight and randomly assigned to one of four treatment groups (n = 11): (i) nonmedicated feed and no MDR Salmonella inoculation (−CTC/−SAL), (ii) medicated feed and no MDR Salmonella inoculation (+CTC/−SAL), (iii) nonmedicated feed and MDR Salmonella inoculation (−CTC/+SAL), and (iv) medicated feed and MDR Salmonella inoculation (+CTC/+SAL). Five days prior to inoculation with SB 403 (−5 dpi), pigs in the +CTC/−SAL and +CTC/+SAL groups began receiving feed supplemented with 400 g of chlortetracycline/ton of feed. Chlortetracycline administration continued following Salmonella challenge throughout the 12-day study.
Pigs in the −CTC/+SAL and +CTC/+SAL groups were inoculated intranasally with 1 ml phosphate-buffered saline (PBS) containing 1 × 109 CFU of strain SB 403 at 0 dpi, as described by Bearson et al. (41). The intranasal route is a natural means for pigs to become infected with Salmonella spp. due to their sniffing and rooting behavior, which brings their nose into contact with other pigs, feces, feed, water, etc. (20). Fecal samples were collected from each pig at −5, −2, 0, 1, 2, 4, and 7 dpi. At 7 dpi, all pigs were euthanized and necropsied, and the following samples were obtained: tonsils, distal ileal mucosa (Peyer’s patch region), ICLN, cecal contents, and cecal mucosa (Fig. 5). Pigs were weighed at −5, 0, and 7 dpi, and rectal temperatures were recorded and fecal moisture content assessed at −5, 0, 1, 2, 4, and 7 dpi, as per Shippy et al. (42). Fecal samples were also collected from the sows just prior to farrowing. Pigs had ad libitum access to feed and water throughout the study. The NADC Animal Care and Use Committee approved all procedures involving animals in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.
Experimental design and timeline of sampling. Pigs in the +CTC groups received in-feed chlortetracycline (CTC) at 400 g/ton of feed beginning at –5 days postinoculation (dpi), and pigs in the +SAL groups were inoculated intranasally with Salmonella Typhimurium SB 403 at 0 dpi. All pigs were necropsied at 7 dpi and ileal mucosal, ileocecal lymph node, cecal contents, cecal mucosal, and tonsil samples obtained. PBS, phosphate-buffered saline. n = 11 for each treatment group.
Bacteriology, gamma interferon, and white blood cell analysis.Blood samples were obtained from the jugular vein at −5, 0, 2, and 7 dpi, and concentrations of white blood cell (WBC), lymphocytes, monocyte, neutrophils, and band neutrophils were determined by the Iowa State University Veterinary Diagnostic Laboratory (Ames, IA). Determination of IFN-γ levels in swine serum of −CTC/+SAL and +CTC/+SAL groups was performed using the porcine IFN-γ enzyme-linked immunosorbent assay (ELISA) kit (Pierce Biotechnology, Rockford, IL, USA), as indicated by the manufacturer’s instructions. Salmonella spp. in the fecal samples of pigs in the −CTC/+SAL and +CTC/+SAL groups were enumerated at 1, 2, 4, and 7 dpi, as detailed by Bearson et al. (25). Briefly, 1 g feces was mixed with 5 ml PBS and serially diluted, and 100 μl was spread onto xylose-lysine-tergitol 4 (XLT4) agar (Becton, Dickinson and Co., Sparks, MD, USA) containing 30 μg/ml nalidixic acid (Becton, Dickinson and Co.). Similarly, 1 g of each tissue sample was placed in a Whirl-Pak bag with 2 ml PBS, pounded with a mallet, homogenized in a stomacher (Seward, Westbury, NY, USA) for 1 min, and serially diluted, and 100 μl was spread onto XLT4 with nalidixic acid. After 48 h of incubation at 37°C, black colonies were enumerated. Samples that were negative for Salmonella spp. by direct plating were qualitatively assessed by enrichment for the presence of Salmonella spp., as previously described (25). Presumptive Salmonella colonies (black) were confirmed using BBL CHROMagar Salmonella agar (Becton, Dickinson and Co.).
Microbial DNA extraction.Fecal, cecal contents, and tonsil samples were placed on ice immediately after collection, transported to the laboratory, and stored at –80°C until DNA extraction. For both the ileal and cecal mucosal samples, DNA from mucosa-associated bacteria was extracted following published protocols (43, 44). Briefly, cecal and ileum sections (10 cm in length) were opened longitudinally and washed several times with sterile PBS to remove digesta and loosely adherent microorganisms and cut in half lengthwise, and each piece was immediately placed in a 50-ml conical tube on ice. Following transport to the laboratory, two 5-mm metal beads and 20 ml of PBS with 0.1% Tween 80 were added to each tube and manually agitated for 1 min. The supernatant was transferred to a sterile 50-ml conical tube and centrifuged at 5,000 × g for 30 min at 4°C. The supernatant was discarded and the pellet retained. An additional 8 ml of PBS with 0.1% Tween 80 was added to each tube containing the cecal or ileal tissue and metal beads, followed by further agitation for 1 min. The supernatant was transferred to the pellet, and the process was repeated once more with the tissue, 8 ml of PBS–0.1% Tween, and beads. The pellet with the supernatant was subsequently centrifuged at 5,000 × g for 30 min at 4°C. Finally, the resulting pellet was snap-frozen with liquid nitrogen and stored at –80°C until DNA extraction.
DNA was extracted from the cecal contents, feces, and pelleted ileal and cecal mucosal samples using the PowerMag microbiome RNA/DNA isolation kit (Qiagen, Germantown, MD, USA), according to the manufacturer’s instructions, and adapted for use on a Biomek FXP laboratory automation workstation (Beckman Coulter, Indianapolis, IN). The DNeasy PowerSoil kit (Qiagen) was used to extract DNA from the tonsil tissue. A bead-beating step of 2 min at 4.0 m/s on a FastPrep FP120 device (Thermo Fisher Scientific, Waltham, MA, USA) was also included after the addition of solution C1. Due to the relatively smaller amount of microbial DNA in the tonsil samples, it was necessary to remove host DNA prior to library preparation. Microbial DNA enrichment was carried out using the NEBNext microbiome DNA enrichment kit (New England BioLabs, Ipswich, MA), as per the manufacturer’s protocol, and the enriched DNA was purified using standard ethanol precipitation (45).
16S rRNA gene sequencing and analysis.The V4 region of the 16S rRNA gene was amplified using the dual-indexing method with the primers 515F (5ʹ-GTGCCAGCMGCCGCGGTAA-3ʹ) and 806R (5ʹ-GGACTACHVGGGTWTCTAAT-3ʹ), as described by Kozich et al. (46). A mock community containing 1 archaeal and 19 bacterial species (47), as well as negative extraction controls, were also included for sequencing. The 16S rRNA gene libraries were sequenced on a MiSeq platform (Illumina, San Diego, CA, USA) using the MiSeq reagent kit version 2 (500 cycles). The 16S rRNA gene sequences were processed using the Dada2 version 1.6 package (48) in R version 3.4.3, with forward reads truncated at 230 bp and reverse reads at 160 bp. A maximum expected error of 2 was used for both paired reads, with no ambiguous bases allowed. Reads were merged and chimeric sequences removed. Taxonomy was assigned to the merged sequences, referred to here as operational taxonomic units (OTUs) at 100% similarity, using the naive Bayesian RDP classifier (49) and the SILVA SSU database release 128 (50), with a 50% bootstrap confidence threshold. QIIME version 1.9.1 (51) was used to calculate the Shannon diversity index and OTU richness, and the R packages vegan version 2.4-6 (52) and Phyloseq version 1.22.3 (53) were used to calculate the Bray-Curtis dissimilarities. OTUs that were predominantly found in the negative extraction controls were removed prior to analysis.
RNA extraction and analysis of the tonsil transcriptome.Tonsil tissue samples were immediately placed in RNAlater (Ambion, Inc., Austin, TX, USA) following removal and stored at –80°C for subsequent RNA extraction. Total RNA was extracted from the tonsil tissue using the miRNeasy minikit (Qiagen), as per the manufacturer’s instructions. RNA quality and quantity were analyzed on a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). The extracted RNA was converted into cDNA and libraries were constructed using the QuantSeq 3ʹ mRNA-Seq forward library prep kit (Lexogen, Greenland, NH, USA), according to the manufacturer’s instructions (54). These single-end libraries were sequenced on an Illumina HiSeq 3000 instrument (150 cycles). The cDNA sequences were trimmed using bbduk version 37.02 (55) and aligned to the Sus scrofa 11.1 genome assembly using STAR version 2.5.2b (56). HTSeq version 0.9.1 (57) was used to count the number of reads that mapped to each gene in the pig genome.
Statistical analysis.A linear mixed model implemented in R version 3.4.3 using lme4 version 1.1.15 (58) was used to assess the fecal and blood parameters, Salmonella concentrations in feces, number of OTUs, and Shannon diversity index by sampling time and treatment group, with individual animal as the random effect. Tukey’s honestly significant difference test (59) was used to perform post hoc comparisons within each sampling time. Salmonella tissue colonization was compared by antibiotic treatment at 7 dpi using an unpaired t test for each tissue type. IFN-γ analysis was performed using a paired t test within a treatment group and an unpaired t test between groups. Contingency analysis of the number of pigs positive for Salmonella tonsil colonization was performed using Fisher’s exact test. Microbial community structures were analyzed with vegan using permutational multivariate analysis of variance (PERMANOVA; adonis function) of Bray-Curtis dissimilarities with 10,000 permutations. Prior to the calculation of diversity metrics and Bray-Curtis dissimilarities, the numbers of reads per sample were randomly subsampled to 3,900, 1,900, 15,500, 12,500, and 5,200 for the tonsil, ileal mucosal, cecal contents, cecal mucosa, and fecal samples, respectively. Differentially abundant OTUs were identified using DESeq2 version 1.18.0 (60) (FDR < 0.05). Only those OTUs present in at least 25% of the samples analyzed were included. DESeq2 was also used to determine differentially expressed genes in the tonsils (FDR < 0.10).
Data availability.All 16S rRNA gene sequences and QuantSeq reads were submitted to the Sequence Read Archive under BioProject number SRP152970.
ACKNOWLEDGMENTS
We are grateful for the outstanding technical support of Briony Atkinson, Jennifer Jones, Margaret Walker, and Kellie Winter, as well as the instrumental assistance from the animal care staff. We thank David Alt for help with sequencing and Julian Trachsel and Darrell Bayles for bioinformatics assistance.
This research was supported by the USDA, Agricultural Research Service (ARS) CRIS funds and National Pork Board grant 16-123. This research used resources provided by the SciNet project of the USDA ARS, ARS project number 0500-00093-001-00-D.
The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendations or endorsement by the U.S. Department of Agriculture.
FOOTNOTES
- Received 26 September 2018.
- Accepted 28 November 2018.
- Accepted manuscript posted online 7 December 2018.
Supplemental material for this article may be found at https://doi.org/10.1128/AEM.02354-18.
- This is a work of the U.S. Government and is not subject to copyright protection in the United States. Foreign copyrights may apply.
